This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cancer cells growing in vivo are very different from cells growing on plastic and respond differently to chemotherapeutic drugs. The objective of this project is to characterize the alterations of the malignant phenotype and drug resistance of bladder cancer calls growing in a more natural, but controlled environment, in extracellular matrix 3D cultures developed in Dr. Hurst's laboratory. The hypothesis of this project is that a "top-down" investigation involving the entire genome in a microenvironment that better replicates in vivo tumors will identify targets and mechanisms to abrogate drug resistance. The long term goal is to identify the signaling networks involved in the drug resistance of a prototypical cancer associated with significant drug resistance, bladder cancer, using cells growing under conditions representative of those found in vivo. With half the genome being poorly annotated, involving the entire genome approach we seek to identify previously non-annotated genes as part of networks ultimately feeding in to novel and known drug resistance pathways. This large-scale picture of drug resistance will identify gene targets for optimization of drug therapy of bladder cancer and other cancers as well as laying out a "big picture" that can focus hypothesis-driven research into new and fruitful areas of research. This will be achieved by a novel bioinformatic approach based on measuring biological variability in the expression of genes under conditions that elicit resistance. Aim 1 analyzes these genes individually as well as identifying functional interconnections between groups of genes playing key roles in drug resistance of cancer cells. Aim 2 seeks to develop a genome-wide functional fingerprint of drug resistance in bladder cancer cells based on synchronized hyper-variable expression of genes. Aim 3 seeks to test the functional roles of gene products in hypothesized epistatic interactions of drug resistance and to refine the models in aims 1 and 2 for the development of a panel of potential targets for drug resistance regulation in bladder cancer. Experimentally validated characteristic changes in gene expression and epistatic interactions resulting in significantly altered serum protein expression will be used to develop a list of potential targets for development of effective therapy of bladder cancer.